Malaria is by far the world's most important tropical parasitic disease, killing more people than any other communicable disease, with the exception of tuberculosis. The causative agents in humans are four species of Plasmodium protozoa: P. falciparum, P. vivax, P. ovale and P. malariae. Although P. falciparum accounts for the majority of infections and is responsible for the vast majority of deaths attributable to malaria, P. vivax causes a recurring chronic debilitating disease for which a vaccine is necessary.
Malaria infection begins when a female Anopheles mosquito infected with one of the four Plasmodium species infectious for humans bites a person. The mosquito's saliva carries the malarial sporozoites into the blood. Approximately 30 minutes later these sporozoites enter the liver. Once in the liver, the sporozoites divide over the course of about 5 days, forming a schizont. A schizont may contain up to 30,000 merozoites, which spill into the bloodstream when the schizont ruptures. Within seconds, merozoites infect red blood cells (RBCs) and again replicate asexually, with each schizont producing up to 36 merozoites.
Each time a RBC bursts and liberates progeny, other blood cells are infected. The cycle can continue until the person dies of anemia and/or other complications. A few of the merozoites in RBCs differentiate into gametocytes, a sexual form, which, if ingested by a mosquito, are liberated from the RBCs in the mosquito stomach and subsequently mate. The progeny, sporozoites, accumulate in the saliva and the process starts again when the mosquito feeds [See, Hoffman et al., (1996) “Attacking the Infected Hepatocyte”, in Malaria Vaccine Development (ed. S. L. Hoffman), p. 35. ASM Press, Washington, D.C., for review).
P. vivax malaria is most prevalent in Latin America (where it is as or more prevalent than P. falciparum) and Asia. Although rarely fatal, P. vivax malaria has a dormant liver phase that is associated with relapses that show variability in duration, depending on the strain.
Presently, there is not an effective vaccine against any form of malaria. For many years, chloroquine was a cheap and effective therapeutic for treating malaria. But in recent years, chloroquine resistance has increased dramatically for P. falciparum. In the past 10 years, P. vivax has also developed chloroquine resistance with cases being reported in south-east Asia, the south-west Pacific; Burma [Marlar, T., et al., Trans. R. Soc. Trop. Med. Hyg., 1995. 89(3): p. 307-308] perhaps India [Garg et al., Trans. R. Soc Trop. Med. Hyg., 1995. 89(6): p. 656-657],Indonesia [Baird et al., Am. J. Trop. Med. Hyg., 1991, 44(5): p. 547-552; Baird et al., Trans R Soc Trop Med Hyg, 1996, 90(4): p. 409-411; Baird et al., J. Infect. Dis., (1995) 171(6): p. 1678-1682; Murphy et al., Lancet, (1993) 341(8837): p. 96-100; and Schwartz et al., [letter]. N. Engl. J. Med., (1991) 324(13): p. 927] and Papua New Guinea [Rieckmann et al., Lancet, (1989) 2(8673): p. 1183-1184].
Primaquine is the only antimalarial drug that is effective against hyponozoites, which are associated with the dormant phase in the liver responsible for relapses. Different P. vivax strains show differential patterns of relapse; for example, a Korean P. vivax strain has been shown to be 100 percent radically cured by a given primaquine regime (WHO, 1967), whereas the same regime is only 70 percent effective with the Chesson strain [Coatney et al., J. Natl. Malaria Soc., 1962. 9: p. 285-292]. To complicate treatment with primaquine further, reports in the 1970s highlighted primaquine-resistant P. vivax in south-east Asia [Charoenlarp et al., Southeast Asian J. Trop. Med. Public Health, (1973) 4(1): p. 135-137 and Krotoski, [letter]. N. Engl. J. Med., (1980) 303(10): p. 587]; observations have steadily increased in other locations in recent years [Schuurkamp et al., Trans. R. Soc. Trop. Med. Hyg., (1992) 86(2): p. 121-122] suggesting that widespread resistance to primaquine is emerging.
Clearly, the most effective approach to combating malaria is an effective vaccine. As with smallpox, and potentially polio in the near future, a coordinated worldwide vaccination program can result in eradication of communicable human diseases. This may also be achievable for malaria if an effective vaccine can be developed.
There are three recognized anti-parasitic approaches to malaria vaccine development. These are proposed to function by interrupting the parasite's lifecycle at three different stages.
The first and most attractive approach is the pre-erythrocytic vaccine, which aims to block sporozoite entry into the hepatocyte and/or release of merozoites into the blood stream. Immediately following infection, sporozoites migrate to the liver and begin the exoerythrocytic stage of their lifecycle. Successful blocking of hepatocyte entry, or the destruction of infected hepatocytes prior to liberation of merozoites, would prevent the disease, the passage of the parasite on to feeding mosquitoes, and merozoite release and subsequent invasion of red blood cells.
A second approach is to develop an ‘antidisease’ vaccine. The target is the red blood cell stage of the infection, during which the parasite grows at an exponential rate. Also known as ‘asexual blood stage’ vaccines, the merozoite surface protein 1 (MSP-1) and apical membrane antigen 1 (AMA-1) protein have emerged as the two most promising vaccine candidates for intervening at this stage of the disease (See, Good, et al., Anu. Rev. Immunol., (1998) 16: p. 57-87,, for review). This stage is thought to represent a conceptually more difficult target compared with the pre-erythrocytic stage, which is associated with 10-20 sporozoites per mosquito bite, due to the tremendous increase in parasite load once the blood stage is reached.
A third approach, known as the ‘transmission-blocking’ vaccine, would not stop infection or symptoms in the individual. However, it would prevent infection from spreading to others by blocking the lifecycle in the mosquito by inducing antibodies that the mosquito would ingest from the host with its blood meal. This vaccine approach is more attractive as a long-term global solution to eradication of malaria and less attractive to the immediate needs of travelers and the military forces.
In the 1960s, researchers at New York University (NYU) achieved full protection from malaria infection by injecting animals with small numbers of sporozoites from mosquitoes that had previously been irradiated. Later, researchers at the University of Maryland, NYU and Walter Reed Army Institute showed that percent of a group of human volunteers immunized with irradiated sporozoites later resisted exposure to virulent sporozoites [Clyde et al., Am. J. Med. Sci., (1973) 266(6): p. 398-403 and Rieckmann et al., Trans. R. Soc. Trop. Med. Hyg., (1974) 68(3): p. 258-259]. This work confirmed that protective immunity to the sporozoite stage (i.e. the pre-erythrocytic stage) of the malaria parasite could be induced. However, an inability to culture sporozoites in vitro thwarted the possibility of using them as a vaccine.
The strategic development of a synthetic malaria vaccine required the identification of immunodominant, neutralizing malaria epitopes. In 1985, a group at NYU led by Drs. Ruth and Victor Nussenzweig, identified the dominant B cell epitope from the circumsporozoite protein (CS), a major component of the sporozoite surface membrane at the time the parasite enters the bloodstream [Zavala et al., Science, (1985) 228(4706): p. 1436-40]. Antibodies to the repeated epitope were shown to be sporozoite neutralizing by protecting against rodent and human malaria [Nussenzweig et al., Ciba Found. Symp., (1986) 119: p. 150-163]. Antibodies to the CS protein also correlated positively with protection in immunized mice and in naturally infected individuals.
These studies strongly suggest that anti-CS repeat antibodies alone are able to confer protection against malaria infection, provided sufficient antibody titers can be raised. The identification of this epitope therefore enabled, for the first time, the strategic development of synthetic CS-based malaria vaccines. Several malaria vaccine candidates employing different carriers were developed based upon the identification of this epitope. The main focus of malaria vaccine development has been on P. falciparum, and it is widely assumed that information gained from studying P. falciparum extend to other Plasmodium species, including P. vivax. A brief overview of four pre-erythrocytic P. falciparum malaria vaccine candidates is given below.
The (NANP)3 synthetic peptide conjugated to the protein carrier tetanus toxoid (TT) was the first synthetic malaria vaccine to undergo phase I and phase II clinical trials in the late 1980s [Etlinger et al., Immunology, (1988) 64(3): p. 551-558; Etlinger et al., J. Immunol., (1988) 140(2): p. 626-633 and Herrington et al., Nature, (1987) 328(6127): p. 257-259]. TT is widely known to provide powerful T cell help for coupled immunogens. Of the thirty-five vaccinees, the three having the highest titers of anti-sporozoite antibodies were selected for challenge studies. One of the vaccine recipients remained free of parasitaemia at 29 days, whereas the other two did not exhibit asexual stage parasites until 11 days, compared with a mean of 8.5 days for the un-vaccinated control group. Therefore, protection again correlated positively with anti-NANP titers.
The limited effectiveness of this vaccine was attributed to suboptimal levels of anti-NANP antibodies. Attempts to increase dosage were hindered by toxicity of the TT carrier. Further, the lack of parasite-derived determinants capable of priming malaria-specific T cells also likely contributed to the low levels of protection.
Short synthetic peptides often have an in vivo half-life that is too short for them to be effective as prophylactic or therapeutic drugs. Standard approaches for increasing the immunogenicity of peptides is to either couple them to larger carrier proteins, or to assemble them into multimeric structures. In this case, 32 copies of the CS repeat sequence ((NANP)15(NVDP))2 were linked and recombinantly fused to a random 32 amino acid fusion protein 20. [Ballou et al., Lancet, (1987) 1(8545): p. 1277-1281.] This vaccine candidate was called FSV-1.
Following immunization, twelve of the fifteen volunteers developed antibodies that reacted with sporozoites. No patients exhibited adverse reactions to the protein, indicating that the NANP (SEQ ID NO: 184) repeat itself is non-toxic. Of the fifteen patients immunized with 3 doses, six were selected to receive a fourth dose and were then challenged with the malaria parasite. Parasitaemia did not develop in the volunteer with the highest titer of CS antibodies, and parasitaemia was delayed in two of the other five vaccinees.
As with the NANP-TT vaccine discussed above, protection correlated positively with anti-NANP titers. This vaccine was deemed partially successful in that it reconfirmed that humans can be protected by CS protein subunit vaccines. However, the level of protection was not sufficient to warrant larger trials of this particular candidate.
A major shortfall of this vaccine was that it did not provide an efficient source of T cell help. The only individuals who would have received T cell help from this vaccine would be those in whom the CS repeat served as both a B and T helper (Th) cell epitope. However, this sequence is known to be a Th epitope for only a limited number of individuals; i.e. it is highly genetically restricted.
Nardin and coworkers at NYU have been able elicit relatively high titers of anti-CS antibody in a diverse range of genetic backgrounds by combining the NANP repeat epitope with the T cell site identified by Berzofsky and Good [Good et al., Science, (1987) 235(4792): p. 1059-62] in a MAP format [Calvo-Calle et al., J. Immunol., (1993) 150(4): p. 1403-1412]. Using their proprietary ‘universal’ form of the CS T cell epitope, Nardin and co-workers have been able to elicit anti-CS antibodies in all genetic backgrounds tested, suggesting that genetic restriction is alleviated by inclusion of this epitope.
Although MAPs have proven to be excellent research tools, providing valuable insight into immune recognition of the CS protein, there are several intrinsic problems associated with using them in a commercial vaccine. Their commercial utility has yet to be established relative to manufacturing and cost issues. Nevertheless, ongoing human clinical testing of these vaccine candidates will provide very useful information pertaining to the actual anti-NANP titers necessary for protective immunity.
One of the most promising malaria vaccines of recent times utilizes the hepatitis B surface antigen (HBsAg) to deliver CS epitopes, an approach developed by SmithKline Beecham (SKB) that is disclosed in U.S. Pat. No. 5,928,902 that issued on Jul. 27, 1999. That patent inter alia discloses a hybrid protein comprised of all of the C-terminal portion of the CS protein, four or more tandem repeats of the CS immunodominant region and the hepatitis B surface antigen. The CS epitopes include the NANP repeat, in concert with additional CS epitopes, including the T cell site identified by Berzofsky and Good [Good et al., Science, (1987) 235(4792): p. 1059-62] (but not the universal form developed by Nardin and co-worker [Moreno et al., Int. Immunol., (1991) 3(10): p. 997-1003 and Calvo-Calle et al., J. Immunol., (1997) 159(3): p. 1362-1373]), fused to the hepatitis B surface protein.
This vaccine was recently the subject of human clinical trials [Stoute et al., N. Engl. J. Med., [1997] 336(2): p. 86-91]. When administered with one of three different adjuvants, this vaccine protected 1/7, 2/7 and 6/7 individuals, respectively. Of the seven individuals immunized with vaccine 2 (adjuvant: oil-in-water emulsion), none of the five patients with anti-CS titers (IFA) in the range of 100-12,800 were protected, whereas the two vaccine recipients with antibody titers in the range of 25,600-51,200 were both protected. Again, protection was correlated positively with anti-CS titers. It is interesting to note that the one patient that received vaccine 1, the alum/oil-in-water formulation, remained protected for at least six months.
The preliminary efficacy report for SKB's malaria vaccine candidate (RTS,S) [Stoute et al. (1997) N. Engl. J. Med., 336(2): p. 86-91] although encouraging, was tempered by the lack of long-term protection in follow-up studies [Stoute et al. (October 1998) J. infect Dis., 178(4):1139-44]. It was also apparent that the use of a potent and complex adjuvant (SBAS2) containing the immunostimulants QS-21 and monophosphoryl lipid A (MPL), formulated in an oil-in-water emulsion, was essential to achieve efficacy, because volunteers receiving the vaccine formulated on alum were not protected. Five of six patients, who were initially protected after administration of the RTS,S/SBAS2 formulation, were not protected six months after receiving the third vaccine dose. Similar results were recently reported at the 48th annual meeting of the American Society of Tropical Medicine and Hygiene for a field trial conducted in Africa.
Like HBsAg, the hepatitis B core antigen (HBcAg), is a particulate protein derived from the hepatitis B virus that has been proposed as a carrier for heterologous epitopes. The relative immunogenicity of HBsAg (HBs) has been compared with HBcAg (HBc), and the ability of each to evoke immune responses in different genetic backgrounds [Milich et al., Science, (1986) 234 (4782) p. 1398-1401]. These data emphasize the higher immunogenicity of HBc relative to HBs, and the universal responsiveness to HBc, irrespective of genetic background.
For example, HBc is more than 300 times more immunogenic than HBs in BALB/c mice; and, although both B10.S and B10.M mice are non-responders to HBs, every strain tested is responsive to HBc. These results re-emphasize the suitability of HBc as a vaccine carrier and specifically, its superiority over HBs, hence the selection of HBc as opposed to HBs to carry heterologous epitopes. These facets of HBc are thought to be particularly important in malaria vaccine development, because they address the genetic restriction and inadequate antibody titers that have been largely responsible for the inability to develop an effective vaccine using the neutralizing CS epitopes.
The positive correlation between protection against malaria infection and anti-CS antibody titer has been demonstrated repeatedly over the past 15 years [Etlinger et al., Immunology, (1988) 64(3): p. 551-558; Etlinger et al., J. Immunol., (1988) 140(2): p. 626-633; Ballou et al., Lancet, (1987) 1(8545): p. 1277-1281; Stoute et al., N. Engl. J. Med., (1997) 336(2): p. 86-91 and Herrington et al.,. Am J Trop Med Hyg, (1991) 45(6): p. 695-701]. The evidence that a vaccine eliciting high-titer, long-lived antibody responses in sufficient vaccine recipients can be protective suggests that protection against malaria infection is achievable via anti-sporozoite antibody production.
Using rodent models of malaria, it has been found that malaria CS-repeats fused to the immunodominant loop of HBc were able to protect mice against both P. berghei and, perhaps more impressively, P. yoelii to levels of 90-100 percent [Schodel et al., Behring Inst. Mitt., 1997(98): p. 114-119 and Schodel et al., J. Exp. Med., (1994) 180(3): p. 1037-46]. Further, antibody responses to the P. berghei particle were shown to prime antibody responses effectively over a wide range of genetic backgrounds, confirming the universal priming effects of HBc [Schodel et al., J. Exp. Med., (1994) 180(3): p. 1037-46].
Another advantage of the HBc carrier is the fact that it does not require complex adjuvants for efficacy. This is due to the high inherent immunogenicity of the particle. A comparison of the immunogenicity of HBc-P. berghei particles showed that alum, which is approved for human use, was more effective than either IFA or CFA [Schodel et al., J. Exp. Med., (1994) 180(3): p. 1037-46]. The importance of this observation is highlighted by toxicity problems associated with newer, more complex adjuvants as was recently noted in clinical trials of SKB's candidate malaria vaccine [Stoute et al., N. Engl. J. Med., [1997] 336(2): p. 86-91].
The immunodominant B cell epitope of the CS protein of P. falciparum, which has been more widely studied than P. vivax, is a highly conserved repeated tetrapeptide (NANP) [Zavala et al., Science, (1985) 228(4706): p. 1436-40], and antibodies to this epitope have been shown to be sporozoite-neutralizing in protecting against rodent and human malaria. Immune responsiveness to this epitope has been positively correlated with immunity to malaria in both vaccine recipients and naturally infected individuals. Indeed, a review of clinical trials data for pre-erythrocytic vaccines described previously (HBs-CS, FSV-1, NANP-TT), highlights a strong correlation between antibody titer and protection. Those individuals who have been protected by previous vaccine candidates have been associated with the highest anti-NANP antibody titers, with the possible exception of SKB's candidate vaccine (#3-RTS,S and adjuvant SBAS2 containing MPL and QS-21 in a water-in-oil formulation) where adjuvants appeared to play a critical role in protection, because protection was not long-lived, as noted before.
The family hepadnaviridae are enveloped DNA-containing animal viruses that can cause hepatitis B in humans (HBV). The hepadnavirus family includes hepatitis B viruses of other mammals, e.g., woodchuck (WHV), and ground squirrel (GSHV), and avian viruses found in ducks (DHV) and herons (HeHV). Hepatitis B virus (HBV) used herein refers to a member of the family hepadnaviridae, unless the discussion is referring to a specific example.
The nucleocapsid or core of the mammalian hepatitis B virus (HBV or hepadnavirus) contains a sequence of 183 or 185 amino acid residues, depending on viral subtype, whereas the duck virus capsid contains 262 amino acid residues. Hepatitis B core protein monomers self-assemble into stable aggregates known as hepatitis B core protein particles (HBc particles). Two three-dimensional structures are reported for HBc particles. A first that comprises a minor population contains 90 copies of the HBc subunit protein as dimers or 180 individual monomeric proteins, and a second, major population that contains 120 copies of the HBc subunit protein as dimers or 240 individual monomeric proteins. These particles are referred to as T=4 or T=3 particles, respectively, wherein “T” is the triangulation number. These human HBc particles are about are about 30 or 34 nm in diameter, respectively. Pumpens et al., (1995) Intervirology, 38:63-74; and Metzger et al., (1998) J. Gen. Viol., 79:587-590. See also, Wynne et al., (June 1999) Mol. Cell, 3:771-780.
Conway et al., (1997) Nature, 386:91-94, describe the structure of human HBc particles at 9 Ångstrom resolution, as determined from cryo-electron micrographs. Bottcher et al. (1997), Nature, 386:88-91, describe the polypeptide folding for the human HBc monomers, and provide an approximate numbering scheme for the amino acid residues at which alpha helical regions and their linking loop regions form. Zheng et al. (1992), J. Biol. Chem., 267(13):9422-9429 report that core particle formation is not dependent upon the arginine-rich C-terminal domain, the binding of nucleic acids or the formation of disulfide bonds based on their study of mutant proteins lacking one or more cysteines and others' work with C-terminal-truncated proteins [Birnbaum et al., (1990) J. Virol. 64, 3319-3330].
The nucleocapsid or viral core protein (HBc) has been disclosed as an immunogenic carrier moiety that stimulates the T cell response of an immunized host animal. See, for example, U.S. Pat. Nos. 4,818,527, 4,882,145 and 5,143,726. A particularly useful application of this carrier is its ability to present foreign or heterologous B cell epitopes at the site of the immunodominant loop that is present at about residue positions 70-90, and more usually recited as about positions 75 through 85 from the amino-terminus (N-terminus) of the protein. Clarke et al. (1991) F. Brown et al. eds., Vaccines 91, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 313-318.
During viral replication, HBV nucleocapsids associate with the viral RNA pre-genome, the viral reverse transcriptase (Pol), and the terminal protein (derived from Pol) to form replication competent cores. The association between the nucleocapsid and the viral RNA pre-genome is mediated via an arginine-rich domain at the carboxyl-terminus (C-terminus). When expressed in heterologous expression systems, such as E. coli where viral RNA pre-genome is absent, the protamine-like C-terminus; i.e., residues at positions 150 through 183, binds E. coli RNA.
In an application as a vaccine carrier moiety, it is preferable that the HBV nucleocapsids not bind nucleic acid derived from the host. Birnbaum et al., (1990) J. Virol. 64, 3319-3330 showed that the protamine-like C-terminal domain of HBV nucleocapsids could be deleted without interfering with the protein's ability to assemble into virus-like particles. It is thus reported that proteins truncated to about position 144; i.e., containing the HBc sequence from position one through about 144, can self-assemble, whereas deletions beyond residue 139 abrogate capsid assembly [Seifer et al., (1995) Intervirology, 38:47-62].
More recently, Metzger et al., (1998) J. Gen. Viol., 79:587-590 reported that the proline at position 138 (Pro-138 or P138) of the human sequence is required for particle formation. Those authors also reported that assembly capability of particles truncated at the carboxy-terminus to lengths of 142 and 140 residues was affected, with assembly capability being completely lost with truncations resulting in lengths of 139 and 137 residues.
Several groups have shown that truncated particles exhibit reduced stability relative to standard hepatitis B core particles [Gallina et al. (1989) J. Virol., 63:4645-4652; Inada, et al. (1989) Virus Res., 14:27-48], evident by variability in particle sizes and the presence of particle fragments in purified preparations [Maassen et al., (1994) Arch. Virol., 135:131-142]. Thus, prior to the report of Metzger et al., above, Pumpens et al., (1995) Intervirology, 38:63-74 summarized the literature reports by stating that the carboxy-terminal border for HBc sequences required for self-assembly was located between amino acid residues 139 and 144, and that the first two or three amino-terminal residues could be replaced by other sequences, but elimination of four or eleven amino-terminal residues resulted in the complete disappearance of chimeric protein in transformed E. coli cells.
Recombinantly-produced hybrid particles bearing internal insertions (referred to in the art as HBc chimeric particles or HBc chimers) often appear to have a less ordered structure, when analyzed by electron microscopy, compared to particles that lack heterologous epitopes [Schodel et al., (1994) J. Exp. Med., 180:1037-1046]. In some cases the insertion of heterologous epitopes into C-terminally truncated HBc particles has such a dramatic destabilizing affect that hybrid particles cannot be recovered following heterologous expression [Schodel et al. (1994) Infect. Immunol., 62:1669-1676]. Thus, many chimeric HBc particles are so unstable that they fall apart during purification to such an extent that they are unrecoverable or they show very poor stability characteristics, making them problematic for vaccine development.
Chimeric hepatitis B core particles have been prepared by heterologous expression in a wide variety of organisms, including E. coli, B. subtilis, Vaccinia, Salmonella typhimurium, Saccharomyces cerevisiae. See, for example Pumpens et al., (1995) Intervirology, 38:63-74 , and the citations therein that note the work of several research groups, other than the present inventors.
A structural feature whereby the stability of full-length HBc particles could be retained, while abrogating the nucleic acid binding ability of full-length HBc particles, would be highly beneficial in vaccine development using the hepadnaviral nucleocapsid delivery system. Indeed, Ulrich et al. in their recent review of the use of HBc chimers as carriers for foreign epitopes [Adv. Virus Res., vol. 50 (1998) Academic Press pages 141-182] note three potential problems to be solved for use of those chimers in human vaccines. A first potential problem is the inadvertent transfer of nucleic acids in a chimer vaccine to an immunized host. A second potential problem is interference from preexisting immunity to HBc. A third possible problem relates to the requirement of reproducible preparation of intact chimer particles that can also withstand long-term storage.
Initial evaluation of a particle displaying epitopes from P. falciparum [CS-2; Schodel et al., J. Exp. Med., (1994) 180(3): p. 1037-46] was encouraging. However, using that particle as an immunogen in a vaccine in mice, provided antibody titers that were lower than those observed for the P. berghei and P. yoelii particles.
There are recognized to be two main CS-repeat epitopes associated with P. vivax (type-I and type-II), and a third, reported in 1993 and called ‘vivax-like’, which is identical to the CS-repeat from the monkey parasite (P. siminovale) resembling P. ovale [Qari et al., Lancet, 1993. 341(8848): p. 780-783]. For simplicity, this CS-repeat is referred to herein as P. vivax type-III.
The benefits of the inclusion of a universal T (Th) cell epitope derived from the malaria parasite are several-fold. First, the priming of malaria-specific Th cells ensures that, should a vaccine recipient be exposed to malaria, a more rapid and stronger anti-malaria response is activated due to previous priming of malaria specific T-helper cells. Secondly, vaccinees living in malaria endemic regions experience natural ‘boosting’ every time they are exposed to the parasite, because their immune systems have been primed at both the B and Th cell level. This effect is similar to clinical boosting by re-vaccination, a process that can be difficult to coordinate in developing countries where malaria is endemic.
Although the CS gene is largely invariant, limited sequence variation has been noted to occur mainly in the immunodominant T cell epitopic domains. The fact that genetic mutations always appear to result in amino acid substitutions suggests that pressure at the protein level, possibly immunological pressure, has selected for variation. Typically, the problems associated with amino acid variability of an epitope can only be resolved by the inclusion of multiple variants of the epitope. However, Nardin and coworkers at New York University recently identified a consensus form of the T cell epitope CS 326-345 that appears to bind all class II MHC molecules [Calvo-Calle et al., J. Immunol., (1993) 150(4): p. 1403-1412 and Moreno et al., J. Immunol., (1993) 151(1): p. 489-499].
Studies have shown that this consensus epitope is ‘universal’, like the T cell help afforded by HBc, and suggests that it primes malaria-specific Th cells in essentially all vaccine recipients. The fact that this epitope of the CS protein was identified by CD4+ T cells of volunteers that were protected against malaria following exposure to irradiated sporozoites, confirms that it is efficiently processed and presented in vivo by antigen presenting cells (APC) when presented in the context of sporozoite [Moreno et al., Int. Immunol., (1991) 3(10): p. 997-1003]. The identification of this epitope was a significant advancement in the task of developing a pre-erythrocytic stage malaria vaccine.
As disclosed hereinafter, the present invention provides a contemplated HBc chimer that provides unexpectedly high titers of antibodies against malaria sporozoites, and in one aspect also provides a solution to the problems of HBc chimer stability as well as the substantial absence of nucleic acid binding ability of the construct. In addition, a contemplated recombinant chimer exhibits minimal, if any, antigenicity toward preexisting anti-HBc antibodies.